A general system for generating unlabelled gene replacements in bacterial chromosomes

Abstract

A general system for generating unlabelled gene replacements in bacterial chromosomes Leenhouts, K.; Buist, Girbe; Bolhuis, A.; Berge, A. ten; Kiel, Jan; Mierau, I.; Dabrowska, M.; Venema, G.; Kok, Jan IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date: 1996Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Leenhouts, K., Buist, G., Bolhuis, A., Berge, A. T., Kiel, J., Mierau, I., ... Kok, J. (1996). A general system for generating unlabelled gene replacements in bacterial chromosomes. Molecular and General Genetics MGG, 253(1-2), 217-224. DOI: 10.1007/s004380050315 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Abstract A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using¸actococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

Genetic characterization of the conjugative DNA processing system of enterococcal plasmid pCF10

“…The improved efficiency of conjugation over electroporation will be particularly helpful in the delivery of suicide vectors for mutagenesis of gram positive bacteria. Allelic exchange in gram positive bacteria often requires a two-step process (Biswas et al, 1993;Leenhouts et al, 1996). The first step involves selection of plasmid transformants under conditions that permit replication of the plasmid.…”

“…Escherichia coli DH5α (Gibco-BRL) and E. coli EC1000, a strain that expresses the pWV01 RepA protein (Leenhouts et al, 1996), were used in cloning. All intron insertion experiments were conducted in E. faecalis.…”

“…pCF10ΔoriT-Fifty-four nucleotides in the pcfE-pcfF intergenic region (pCF10 nucleotides 32,812-32,866) were deleted and replaced with a PstI site (CTGCAG), using an improved allelic exchange system recently described in detail (Kristich et al, 2005). DNA from upstream and downstream of the region to be deleted was PCR amplified and cloned into pCJK2, a pORI280 (Leenhouts et al, 1996) derivative. Primers used to amplify the upstream fragment incorporated XmaI and PstI restriction enzyme sites while primers used to amplify the downstream fragment incorporated PstI and XbaI sites, simplifying stepwise cloning into pCJK2.…”

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“…07. GSM17E 5 agar medium: as in 3 but add 10 g [14] 01. Insert two flanking fragments of the region to be deleted in the multiple cloning site (mcs) of pORI280 and transform to E. coli EC1000 (RepA + ).…”

“…Figure 1 summarises the characteristics of the Ori + -system. The pWV01 repA gene under the control of the lactococcal promoter P 23 , which is expressed in Gram-positive as well as in Gram-negative bacteria [30], was integrated into the chromosomes of strains of E. coli, B. subtilis and L. lactis [10,[13][14][15]. These RepA + strains produce the RepA protein in trans, thus sustaining the replication of pWV01-derivatives lacking repA, but still carrying the recognition site for the initiation of replication (pORI plasmids).…”